Preparation of a cardio-active glycoside



Patented Oct. 28, 1952 PREPARATION OF A CARDIO-ACTI'VE GLYCOSIDE Harry Rosen, Delaware County, and Casimir T.

Ichniowski, Philadelphia County, Pa., assignors to Wyeth Incorporated, Philadelphia, Pa., a

corporation of Delaware No Drawing. Application June 30, 1949, Serial No. 102,436

This invention relates to the preparation of a cardio-active glycoside and more particularly relates to the extraction and isolation of an active glycoside of digitalis in a substantially pure and crystalline state.

This application is a continuation-in-part of application Serial No. 41,905, filed July 31, 1948, now Patent No. 2,557,916.

The above-mentioned prior application and this application deal with a method for obtaining digitoxin (C41H640'13) in a highly purified form, the product being characterized by its even and uniform pharmacological action substantially free of undesirable side reactions.

The process disclosed in said prior application involves, essentially, a series of solvent treatment steps following the extraction of the active principles from digitalis leaves. Briefly, the former process involves, first, the removal of substantially inert material by the use of a solvent which dissolves the inert material, which is then followed by a second solvent extraction procedure wherein the active principles are themselves selectively removed by a substantially water-immiscible organic solvent in which they are soluble. The invention disclosed herein is an improvement over this prior procedure by eliminating the necessity for a two-solvent operation of the type described and requiring merely a single solvent extraction treatment which effectively and selectively removes digitoxin in good yields from substantially inert and undesired material. By eliminating the two-step solvent procedure, it is obvious that less equipment is necessary and in this respect the process is less costly. In addition, less fiuid need be handled and thus one also avoids possible losses due to the use of extra steps.

One object of the present invention is to provide substantially pure, crystalline digitoxin, the chief cardio-active glycoside of digitalis.

Another object of the invention is to extract digitoxin more completely from crude sources, such as the leaves of Digitalis purpurea.

Another object of the invention is to isolate in a substantially pure, crystalline form the digitoxin contained in extract of digitalis.

Other objects will become apparent in the following disclosure.

Briefly describing the invention, digitalis leaves are extracted with an organic solvent and water solution. This extraction removes substantially all of the desired cardio-active glycoside, together with a certain proportion of undesired and inert substances. The extract containing the glycoside 8 Claims. (01. 260-2105) cible organic solvent in which the desired cardioactive principles are selectively soluble to the exclusion of a large portion of the undesired substances. In order to remove the small amount of undesired, inert substances which are unavoidably dissolved in the solvent, the solvent extract is washed with water or a dilute alkaline solution which effectively removes a large part of these impurities. The now purified extract substantially freed of all inert matter is then distilled to remove the solvent. The digitoxin precipitates on concentration and isthen isolated for subsequent further purification and recovery.

In a more preferred procedure, the digitalis leaves are first admixed with a chemical tanninand vegetativeprecipitant, or insolubilizing agent. This material prevents a large part of the undesired substances, such as tannins, resins, gums, etc., from interfering with the extraction of the active principles by either precipitating these undesired substances or rendering them less soluble in the following extractive procedure. There is also the possibility that there is both a precipitating and insolubilizing effect. The admixed leaves and chemical agent are now given the extraction treatment with the water-soluble solvent as indicated hereinbefore.

The chemical agent acting as a tannin and inert material precipitant and insolubilizing agent which is admixed with the digitalis leaves is a compound comprising a bivalent metal such as lead, iron, copper, tin, and chromium in the form of their acetates, chlorides or carbonates or their basic salts. These compounds may be used either in solid form as aqueous suspensions or in solution, at a concentration preferably of about 25% by weight per unit volume. Among the compounds mentioned, ferrous and lead acetate have been foundto be particularly suitable, with the latter more particularly preferred.

The extraction of the digitalis leaves orthe mixture of digitalis leaves and bivalent metal salts may be carried out either by a straight, continuous or fractional percolation with a menstruum, comprising water and a water-soluble organic solvent. As a particularly useful watersoluble solvent may be mentioned dioxane but a more preferred solvent is a lower alcohol of mixture of lower alcohols which extracts the waterinsoluble digitoxin substantially completely from digitalis leaves when admixed with water. .By lower alcohol, the C1-C4 alcohols are meant, particularly methyl, ethyl or isopropyl alcohol. These may be used either singly or in admixture.

' The concentration of Water-soluble solvent in aqueous solution suitable for use ranges from approximately 50% solvent to about 95% solvent, the remainder being water. In general, however, it is preferred that the inenstruum comprises a major amount of solvent and a minor amount of water. As a preferred solvent denatured ethyl alcohol is used when the denaturant is present in minor amount and is such that it would not react with the desired digitoxin; and,more preferably still, because of relative cost, Standard Denatured Alcohol No. 30, a denatured ethyl a1- cohol with methyl alcohol as the denaturant, made up in an ethyl to methyl alcohol ratio by volume of about 1, can-be used. The alcohol is preferably mixed with water on the basis of about 60-70% by volume of alcohol and about -40% water for best extraction of active principles.

Following the extraction or percolation to obtain the active principles in solution, the aqueous solvent extract is now concentrated to the point where-substantially all of the solvent is removed, thus bringing the extract, which is now largely aqueous, to the point where it is ready for extraction of the cardio-active glycoside.

The substantially aqueous extract is new extracted a number of times with an organic, substantially water-immiscible solvent in which the desired cardio-active principles are soluble. It has been discovered that chlorinated solvents are especially suitable for this purpose, although other water-immiscible solvents such as isopropyl ether, dimethyl carbonate and solvents of like nature have been found efiective. It has been found, however, that substantial water-immiscibility is a critical-requirement. By the term water-immiscible or substantially water-immiscible solvent is meant any solvent having a solubility in water of less than about 1 part per 100 under general atmospheric conditions.

A large number of chlorinated compounds have been investigated, and it has been found that the water-immiscible, chlorinated, aliphatic hydrocarbons of two or more carbon atoms are particularly eifective. Chlorinated solvents-having a single carbon atom, such as carbon tetrachloride or methylene chloride, for example, are totally ineffective since digitoxin is either not soluble therein or the solvent decomposes under the conditions of operation. On the other hand,

it is well-known that chloroform has substantial solvent powers but is so indiscriminate and nonselective in its effect by dissolving large quantitles of undesired, inert material together with digitoxin that it is valueless by itself if a pure product is desired. Unsuitable'as'this lattermaterial is by itself, it has been found, however, that its solvent capacity can be effectively utilized if a modifying agent is added to make it selective for the digitoxin principles and reduce or depress its solubilizing effect for the inert materials. Selected ethers, such as diethyl or isopropyl ether for example, are effective modifying agents forchloroform, the mixture then becoming substantially selective in its cardio-active 1 acne, the di-chloro compounds as ethylidene dichloride and propylene dichloride, the tricliloro compounds such as tri-chloro-ethylene, methyl chloroform, etc. These chlorinatedhydrocarbons l iii) not higher than about 100 C. under atmospheric conditions is preferred.

One of the above-mentioned 'solventswhich has been found to be particularly preferable because of its high solvent capacity for the desired cardio-active principle without also dissolving undesirable amounts of inert material and which is relatively low boiling is ethylidene dichloride.

Following the extraction with the water-immiscible 'di gitoxin solvent, the organic solvent extract is now washed a number of times with water or an aqueous alkaline solution 'until'the washes are substantially colorless. This procedure removes practically all of the water-soluble vegetable extractive materials leaving, in soluti'on, the digitoxin in a highly purified state.

When using aqueous "alkaline solutions, alkali metal hydroxides or carbonates-preferably sodium-carbonatemay-be used. An aqueous solution of about 1-13% alkaline material is preferred.

The purified solution of active principles in the water-immiscible solvent is now dried, =filtered, and concentrated to remove a substantial amount of solvent. Precipitation of digi-tox'in takes place when a substantial portion of the solvent has been removed. "With regard to this concentration step or in fact any heating step while the active principles are commingled with crude material and solvent, it is important to watch that the temperature does not go beyond about C. When the product is still in crude form, it has been found that temperatures substantially above this point result in a lowered yield of the desired product. To remove solvent under this limiting temperature obviously the usual, well-known distillation and separatory procedures may be utilized, such-as'vacuum, partial pressure operations, etc. Following the concentration and precipitation steps, the final precipitate may be further purified.

The purification and crystallization of crude digitoxin is carried out'by dissolving the precipitate in ab-out'95% alcohol'at a temperature of about -65 C. and diluting the solution with water at a temperature of about GIL- C.'to an alcohol concentration of about 40%. The solution is permitted to cool and purified digitoxin precipitates therefrom. The product is then filtered on and dried. Additional purifications may be carried out if a highly purified product is desired.

For a better understanding of the invention,

1 specific examples follow, it being clearly understood that these are merely for illustrative purposes only and not to be considered limitative of the invention.

Example I 1000 gm. dried ground digitalis leaves (Digitalis purpurea) are moistened with 250 cc. 25% (w./vl) lead acetate solution. The leaded drug is allowed to stand about one hour, and is then moistened further with 250 cc. 60%

'.(w./v'.) S- D. A. #30 alcohol, and allowed to stand for another hour.

It is then transferred to asuitable percolator and 60% S. D. A. #30

'alcohol is added until the percolate flows from the lower orifice and a stratum of menstruum remains above the drug. The lower orifice is closed and allowed to macerate for a number of hours. After the maceration has been completed, the percolation is then started. The

first 400 cc. of percolate is collected and la- -beled A reserve.

Fraction #2, A Fraction #3, A Fraction #4, through A Fraction #5.

When these five A fractions are obtained, percolation of the A portion of the drug is completed.

The B portion of 600 gm. dried ground digitalis leaves (Digitalis purpurea) is now moistened with 150 cc. 25% lead acetate solution (w./v.). This is permitted to stand about one hour. The leaded drug is now moistened with 150 cc. of A Fraction #1 percolate and also permitted to stand one hour. It is now transferred to a suitable percolator and the remainder of A Fraction #1 percolate is added. As this percolate disappears below the surface of the drug, A Fraction #2 is added. This described procedure is "continued, adding the A fraction percolates in increasing numerical succession, one at a time, until the percolate begins to fiow from the percolator and a stratum of menstruum remains above the drug. The lower orifice of the percolator is closed and the maceration is allowed to continue for a number of hours. Thereafter, the percolation is started and one collects the first 600 cc. This 600 cc. portion is labeled B reserve and is not used until percolation of all portions of the drug is completed. Percolation is continued using as the menstruum the remaining A fractions, adding these, one at a time, in their ascending numerical order. After these A fractions have been used, percolation is continued with 60% S. D. A. #30 alcohol, if necessary, using enough to obtain five successive fractions of percolate of 400 cc. each. These fractions are labeled, respectively, fB Fraction #1, B Fraction #2, B Fraction #3, B Fraction #4, through B Fraction #5. When the B reserve and the five B fractions have been obtained, percolation of the B portion of 600 gm. of ground digitalis has been completed.

The third or last portion of 400 gm. of dried ground digitalis leaves (Digitalis purpurea) is moistened with 100 cc. 25% (w./v.) lead acetate solution. This is permitted to stand for about one hour. The leaded drug is further mois-'.

, and a stratum remains above the drug. The

lower orifice of the percolator is closed and the maceration is allowed to continue for a number of hours. Percolation is now carried out, the percolates being collected until 1000 cc. are ob- 6 tained. This is labeled Percolate C Reserve. As the percolation proceeds in obtaining C reserve, one adds, as needed and one at a time, the remaining B fractions of percolate. On rare occasions, it may be necessary to use a small amount of 60% S. D. A. #30 alcohol in order to obtain the 1000 cc. of percolate. When the C reserve of 1000 cc. has been collected, the

percolation is completed.

The A, B, and C reserves are. combined to make a total of 2000 cc. These 2000 cc. comprise the digitalis percolate to be processed for digitoxin. These combined reserves of 2000 cc. are transferred to a suitable vacuum still and concentrated at or below about 40 C. to about 70-75%.

of the original volume of percolate.

The concentrate is now transferred to a suitable extraction apparatus and is extracted with successive portions of a water-immiscible digitoxin solvent, in this case, ethylidene dichloride. Each portion of ethylidene dichloride is equal to of the volume of this concentrated percolate. Layers form, and the ethylidene dichloride layer is removed at the bottom, the top layer being discarded after the last extraction. Five or six such extractions usually suffice for complete extraction or the concentrate. The ethylidene dichloride extractions are then combined.

This composite of ethylidene dichloride solution is now extracted with 2000 cc. portions of 1% sodium carbonate (w./v.) solution, prepared by using anhydrous sodium carbonate and distilled water. The sodium carbonate extractions are continued until the sodium carbonate solution, after removal from the ethylidene dichloride mixture, exhibits no color when viewed in a 250 cc. filled Erlenmeyer flask. Each sodium carbonate. wash is discarded as it is removed.

The washed ethylidene dichloride solution is transferred to a suitable container and a sufficient quantity of anhydrous sodium sulfate is added to remove all traces of entrained aqueous solution. The dried ethylidene dichloride solution is filtered to remove completely all the sodium sulfate, and is then concentrated in vacuo (about 25-28 inches of Hg vacuum) to about 100 cc. The concentrate is removed from the vacuum still and is allowed to cool to room temperature. A precipitate of crude digitoxin is obtained which is isolated'by filtration. The precipitate is then washed with small portions of ethylidene dichloride and finally with a small quantity of diethyl e her.

To further purify the digitoxin, the precipitate is air-dried and is then dissolved in U. S. P. alcohol at 65 C. using 1 cc. of this 95% alcohol .U. S. P. for 30 mg. of dry solid. This is filtered,

if necessary, at 65 C. The alcoholic solution is now diluted with a suflicient quantity of hot distilled water at 65 C. to make the resulting alcohol concentration approximately 40%. On cooling slowly to room temperature, the crystalline digitoxin separates out which is then separated by filtration and dried to constant weight.

Example II 2; 6:135; 8&4

Example III Example IV The same procedure as given in Example is carried out using n-propy-l chloride as the digitoxin extracting solvent. However, in the extraction procedure, the upper layer, in the two layers obtained, contains the active principles dissolved in the n-propyl chloride and thus the bottom layer is discarded after the last extraction.

Example V The-same procedure as given in Example I was carried out using a mixture of solvents comprising 90% diethyl ether and 10% chloroform. However, as in the case of Example IV, the upper layer contained the active principles and the lower layer was the one that was finally discarded.

Example VI Ground digitalis leaves were admixed with lead acetate and fractionally percolated in the manner described in Example I, using, however, 40% dioxane solution in water in place of the 60% S. D. A. #30 alcohol. From this percolation 2000 cc. of percolate was obtained.

The percolate was extracted with {our extractions of 1000 cc. portions ethylidene dichloride. These extractions were combined and washed with eight 1000 cc. portions of 1.0% sodium carbonate solution. The washed extract was dried with anhydrous sodium sulfate and filtered.

The organic solution was now concentrated in vacuo to a paste. This was then reconstituted with a small amount of ethylidene dichloride sufficient to make a fluid which was filtered, washed and dried. The crude digitoxin was then crystallized by the procedure as described in Example I, using 1.0 cc. of alcohol for each 30 mgm. of crude material. This was diluted with water at 65 C. to an alcohol concentration of 40%. The digitoxin was allowed to crystallize out and was separated and dried.

Example VII In order to carry out a straight percolation rather than the fractional percolation described in Examples I and VI, 1000 grams of ground digitalis' leaves are moistened with 250 cc. of w./v. lead acetate solution. Then, 250 cc. of 40% dioxane solution in water is added. The mixture is permitted to macerate overnight and is percolated, adding 40% dioxane solution as needed until 2500 cc. percolate is obtained. The percolate is then treated in substantially the same manner as described in Example VI.

Example VIII The ground digitalis is treated and packed in a percolator as in Example 1. However, percolation here is continuous rather than fractional.

2 kgms. of digitalis were percolated with 60% S. D. A. until 7200 cc. of percolate were obtained. A 3600 cc. aliquot of thepercolatexwas concentrated in vacuo to 1700 cc. This. comcentrate was extracted with four 600 cc; portions of propylene dichloride. The combined propylene dichloride extracts were washed with nine. 1000 cc. portions of 1% NazCOs. The washed propylene dichloride solution was dried with NazSOa, filtered, and the filter washed with propylene dichloride. This dried solution was concentrated in vacuo to approximately 50 00., let cool to room temperature and then filtered. The crude material was crystallized as described in Example'I.

Example IX The same procedure as given in Example I was carried out using trichloroethylene in place of ethylidene dichloride. As in the case. of the dichloride extraction, the lower, heavier layer*con tains the active principles dissolved in the waterimmiscible solvent.

Example X The same procedure as described in Example I was carried out using isopropyl ether in place of ethylidene dichloride. Having a low density, the ether layer containing the active principles was removed at the top and the bottom layer, after the extractions, was finally discarded. The. yield of digitoxin, while acceptable, was found to be considerably below that obtained with ethylidene dichloride. However, when isopropyl other is mixed with chloroform to the extent of approximately 70% ether and 30% chloroform, by volume, the yield of digitoxin is substantially equal to that obtained with ethylidene dichloride.

Suitable changes and modifications in reagents and ingredients used that do not, depart from the spirit of the disclosure herein, are contemplated as falling within the scope of. the in.- vention.

Having described our claim is:

1. A process for the extraction and isolation of cardio-active glycosides from crude sources comprising moistening the leaves of Digitalis purparea with a tanninand vegetative-insolubilizing agent dissolved in an aqueous medium, extracting the moistened leaves with a solvent comprising a major amount of a lower aliphatic alcohol and a minor amount of water in which the cardio-active principles are soluble, concentrating the resulting first extract to separate a subtantial portion of the alcohol, contacting the aqueous extract with ethylidene dichloride in which the desired cardio-active principles are soluble, washing the ethylidene dichloride solution with an aqueous alkaline solution, separating the washed solution and concentrating to the point where digitoxin precipitates out as the desired product.

2. A process for the extraction and isolationof digitoxin principles from crude sources comprising moistening the leaves of Digitalis purpurea with a tanninand vegetative-insolubilizing agent, extracting the moistened leaves with a solvent of the group consisting of approximately equal amounts of dioxane and water and an alcoholic solvent comprising a. major amount of a lower aliphatic alcohol and a minor amountof water, concentrating the solvent extract, contacting the concentrated extract with a substantially water-immiscible, chlorinated solvent medium selected from the group consisting of propyl chloride, butyl chloride, mono-chlorobenzene, ethylidene dichloride, propylenedichloride, triinvention. what we chloroethylene, methyl chloroform, a mixture of chloroform and diethyl ether and a mixture of chloroform and diisopropyl ether, in which the desired digitoxin principles are selectively soluble while impurities are relatively insoluble, washing the solvent solution with an aqueous alkaline solution, separating the washed solvent solution and concentrating to the point where the digitoxin principles can precipitate out of the solution.

3. The process of claim 2; wherein the tanninand vegetative-insolubi1izing agent is lead acetate in an aqueous medium.

4. A process for the extraction and isolation of digitoxin principles from crude sources comprising moistening the leaves of Digitalis purpurea with a tanninand vegetative-insolubilizing agent, extracting the moistened leaves with an alcoholic solvent comprising a major amount of a lower aliphatic alcohol and a minor amount of water, concentrating the solvent extract, contacting the concentrated extract with a stable, substantially water-immiscible, chlorinated lower aliphatic solvent having at least two carbon atoms in its chemical structure in which the desired digitoxin principles are soluble, washing the solvent solution containing the digitoxin 10 principles with an aqueous alkaline solution, separating the washed solvent solution and concentrating it to the point where the digitoxin principles can precipitate out of the solution.

5. The process of claim 4; wherein the chlorinated lower aliphatic solvent has a solubility in water of not more than about one part per parts of water at atmospheric conditions.

6. The process of claim 4; wherein the stable chlorinated lower aliphatic solvent boils below about 100 C.

'7. The process of claim 4; wherein the chlorinated lower aliphatic solvent is propylene dichloride.

8. The process of claim 4; wherein the chlorinated lower aliphatic solvent is trichloroethylene.

HARRY ROSEN. CASIMIR T. ICHNIOWSKI.

Name Date Number I Rosen Sept. 21, 1948 

1. A PROCESS FOR THE EXTRACTION AND ISOLATION OF CARDIO-ACTIVE GLYCOSIDES FROM CRUDE SOURCES COMPRISING MOISTENING THE LEAVES OF DIGITALIS PURPUREA WITH A TANNIN- AND VEGETATIVE-INSOLUBILIZING AGENT DISSOLVED IN AN AQUEOUS MEDIUM, EXTRACTING THE MOISTENED LEAVES WITH A SOLVENT COMPRISING A MAJOR AMOUNT OF A LOWER ALIPHATIC ALCOHOL AND A MINOR AMOUNT OF WATER IN WHICH THE CARDIO-ACTIVE PRINCIPLES ARE SOLUBLE, CONCENTRATING THE RESULTING FIRST EXTRACT TO SEPARATE A SUBSTANTIAL PORTION OF THE ALCOHOL, CONTACTING THE AQUEOUS EXTRACT WITH ETHYLIDENE DICHLORIDE IN WHICH THE DESIRED CARDIO-ACTIVE PRINCIPLES ARE SOLUBLE, WASHING THE ETHYLIDENE DICHLORIDE SOLUTION WITH AN AQUEOUS ALKALINE SOLUTION, SEPARATING THE WASHED SOLUTION AND CONCENTRATING TO THE POINT WHERE DIGITOXIN PRECIPITATES OUT AS THE DESIRED PRODUCT. 